Enzymes are biological molecules that catalyze a chemical reaction of a substrate. To understand the reaction mechanism and kinetics of such enzymatic reactions, it is often desirable to characterize an enzyme by determining its activity. Enzyme characterizations are carried out with assays that quantitatively assess enzyme activity based on, for example, resulting concentrations of one or more products of the enzymatic reaction. Thus, enzyme characterization provides information about the enzyme activity that can be used to predict how the enzyme will behave when reaction mixtures are altered, such as by adjusting the amounts of substrate, enzyme, etc., involved. Characterizing enzyme activity at different temperatures or pH levels provides information about the enzyme activity that can be used to predict how the enzyme will behave when reaction conditions are altered.
Typically, enzyme activity is determined by measuring the concentration of a reaction product of an enzymatic reaction over time for a fixed and constant enzyme concentration. Thus, the higher a reaction product concentration detected, in a period of time, the higher the enzyme activity determined. However, the reaction product concentration levels may be impacted by conditions other than enzyme activity. For example, some enzymatic reactions are carried out at high temperatures (e.g., at temperatures above about 50° C.) that can cause reagents of the reaction to evaporate, skewing concentration measurements. That is, if the reagents evaporate during the reaction, resulting concentrations of reaction products may be determined to be artificially higher than they would otherwise be, indicating a false-high enzymatic activity, or the evaporation may concentrate the enzyme in the reaction, resulting, again, in artificially high measured concentrations of reaction products. As another example, some substrates are generally insoluble in the reagents, and mass transfer properties, such as the diffusivity of the enzyme into the substrate or the diffusivity of a reaction product out of the substrate, may control the rate of the enzymatic reaction and, thus, the rate of production of the reaction product. Therefore, measured reaction product concentrations may be artificially low based on a low diffusivity value, rather than on the actual rate of the enzymatic reaction. As still another example, not all substrates and enzymes readily intermix with one another. Low intermixing may result in less enzymatic activity and, thus, low reaction product concentrations, even if the reaction rate is actually rapid.
Additionally, conventional methods for enzyme characterization may not be well suited to evaluate the enzymatic activity using substrates that are of industrial relevance. Not only may some industrially-relevant substrates be generally insoluble, but some may additionally or alternatively be generally heterogeneous such that one small sample of the substrate may vary in composition from another small sample of the same substrate. To try to avoid such heterogeneity impacting the results of enzymatic characterization methods, many conventional methods involve the use of a large amount of the substrate. However, use of a large amount of substrate may require use of a large amount of enzyme in the characterization. Such large-volume methods may not be conducive for characterizing enzymes for which only small amounts are available.
Thus, accurately characterizing enzymes for high-temperature (i.e., greater than about 50° C.) reactions, reactions with insoluble substrates, reactions with substrates and enzymes that do not readily intermix, reactions with heterogeneous substrates, or reactions where large quantities of enzymes or substrates are not available often presents challenges.